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Identification of pregnane-X receptor target genes and coactivator and corepressor binding to promoter elements in human hepatocytes.

Hariparsad N, Chu X, Yabut J, Labhart P, Hartley DP, Dai X, Evers R - Nucleic Acids Res. (2009)

Bottom Line: Genes, both previously known to be involved and not known to be involved in drug disposition, with PXR response elements (PXREs) located upstream, within or downstream from their potentially associated genes, were identified.We observed promoter-dependent patterns with respect to the binding of various coactivators and corepressors involved in the regulation of CYP4F12, CYP3A4, CYP2B6, UGT1A1 and P-glycoprotein.In conclusion, our findings indicate that PXR is involved in the regulation of CYP4F12 and that PXR along with SRC1 binds to a broad range of promoters but that many of these are not inducible by rifampicin.

View Article: PubMed Central - PubMed

Affiliation: Department of Drug Metabolism and Pharmacokinetics, Merck & Co, Rahway, NJ 07065, USA. niresh_hariparsad@merck.com

ABSTRACT
Chromatin immunoprecipitation (ChIP) studies were conducted in human hepatocytes treated with rifampicin in order to identify new pregnane-X receptor (PXR) target genes. Genes, both previously known to be involved and not known to be involved in drug disposition, with PXR response elements (PXREs) located upstream, within or downstream from their potentially associated genes, were identified. Validation experiments identified several new drug disposition genes with PXR binding sites. Of these, only CYP4F12 demonstrated increased binding in the presence of rifampicin. The role of PXR in the basal and inductive response of CYP4F12 was confirmed in hepatocytes in which PXR was silenced. We also assessed the association of PXR-coactivators and -corepressors with known and newly identified PXREs. Both PXR and the steroid receptor coactivator (SRC-1) were found to bind to PXREs in the absence of rifampicin, although binding was stronger after rifampicin treatment. We observed promoter-dependent patterns with respect to the binding of various coactivators and corepressors involved in the regulation of CYP4F12, CYP3A4, CYP2B6, UGT1A1 and P-glycoprotein. In conclusion, our findings indicate that PXR is involved in the regulation of CYP4F12 and that PXR along with SRC1 binds to a broad range of promoters but that many of these are not inducible by rifampicin.

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Effect of PXR knockdown on the basal expression and inductive response of CYP4F12, CYP3A4, CYP2B6, MDR1 and UGT1A1 in human hepatocytes. Hepatocytes were transfected with ON-TARGETplus SMARTpool human NR1I2 siRNA (PXR-siRNA) or the non-specific ON-TARGETplus Non-Targeting Pool siRNA (scrambled, SCR-siRNA) as described in the Materials and methods section. (A) The effect of PXR silencing on basal expression of genes was determined relative to hepatocytes transfected with non-specific or SCR-siRNA. (B) The effect of PXR silencing on the inductive response was determined in hepatocytes transfected with PXR-siRNA or non-specific SCR-siRNA treated with rifampicin 10 µM. The error bars represent the SD from the mean of triplicate assays of an individual experiment, n = 3, *P < 0.05.
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Figure 6: Effect of PXR knockdown on the basal expression and inductive response of CYP4F12, CYP3A4, CYP2B6, MDR1 and UGT1A1 in human hepatocytes. Hepatocytes were transfected with ON-TARGETplus SMARTpool human NR1I2 siRNA (PXR-siRNA) or the non-specific ON-TARGETplus Non-Targeting Pool siRNA (scrambled, SCR-siRNA) as described in the Materials and methods section. (A) The effect of PXR silencing on basal expression of genes was determined relative to hepatocytes transfected with non-specific or SCR-siRNA. (B) The effect of PXR silencing on the inductive response was determined in hepatocytes transfected with PXR-siRNA or non-specific SCR-siRNA treated with rifampicin 10 µM. The error bars represent the SD from the mean of triplicate assays of an individual experiment, n = 3, *P < 0.05.

Mentions: We conducted siRNA studies to further discern the involvement of PXR in the regulation of CYP4F12, UGT1A5, ABCB4 and ABCC4 which were identified as having strong PXR binding sites using ChIP analysis. We observed 79% knockdown in PXR mRNA levels in hepatocytes transfected for 72 h with PXR-siRNA relative to hepatocytes that were transfected with SCR-siRNA (data not shown). The basal expression levels of known PXR target genes, such as CYP3A4, CYP2B6, MDR1 and UGT1A1 were significantly lower (54–97%) in hepatocytes in which PXR was knocked down using PXR-siRNA compared to non-specific SCR-siRNA (Figure 6A). A statistically significant (∼80%, P < 0.05) decrease was also observed in the basal expression of CYP4F12. While ∼30% decrease was observed in the basal expression of UGT1A5, this was not statistically significant when compared to hepatocytes transfected with the non-specific SCR-siRNA. To confirm the specificity of the PXR-siRNA we also determined whether basal expression levels of the housekeeping genes 18S and GAPDH were affected. We did not observe a statistically significant decrease in the expression of these genes (data not shown) following PXR-siRNA transfection.Figure 6.


Identification of pregnane-X receptor target genes and coactivator and corepressor binding to promoter elements in human hepatocytes.

Hariparsad N, Chu X, Yabut J, Labhart P, Hartley DP, Dai X, Evers R - Nucleic Acids Res. (2009)

Effect of PXR knockdown on the basal expression and inductive response of CYP4F12, CYP3A4, CYP2B6, MDR1 and UGT1A1 in human hepatocytes. Hepatocytes were transfected with ON-TARGETplus SMARTpool human NR1I2 siRNA (PXR-siRNA) or the non-specific ON-TARGETplus Non-Targeting Pool siRNA (scrambled, SCR-siRNA) as described in the Materials and methods section. (A) The effect of PXR silencing on basal expression of genes was determined relative to hepatocytes transfected with non-specific or SCR-siRNA. (B) The effect of PXR silencing on the inductive response was determined in hepatocytes transfected with PXR-siRNA or non-specific SCR-siRNA treated with rifampicin 10 µM. The error bars represent the SD from the mean of triplicate assays of an individual experiment, n = 3, *P < 0.05.
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Related In: Results  -  Collection

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Figure 6: Effect of PXR knockdown on the basal expression and inductive response of CYP4F12, CYP3A4, CYP2B6, MDR1 and UGT1A1 in human hepatocytes. Hepatocytes were transfected with ON-TARGETplus SMARTpool human NR1I2 siRNA (PXR-siRNA) or the non-specific ON-TARGETplus Non-Targeting Pool siRNA (scrambled, SCR-siRNA) as described in the Materials and methods section. (A) The effect of PXR silencing on basal expression of genes was determined relative to hepatocytes transfected with non-specific or SCR-siRNA. (B) The effect of PXR silencing on the inductive response was determined in hepatocytes transfected with PXR-siRNA or non-specific SCR-siRNA treated with rifampicin 10 µM. The error bars represent the SD from the mean of triplicate assays of an individual experiment, n = 3, *P < 0.05.
Mentions: We conducted siRNA studies to further discern the involvement of PXR in the regulation of CYP4F12, UGT1A5, ABCB4 and ABCC4 which were identified as having strong PXR binding sites using ChIP analysis. We observed 79% knockdown in PXR mRNA levels in hepatocytes transfected for 72 h with PXR-siRNA relative to hepatocytes that were transfected with SCR-siRNA (data not shown). The basal expression levels of known PXR target genes, such as CYP3A4, CYP2B6, MDR1 and UGT1A1 were significantly lower (54–97%) in hepatocytes in which PXR was knocked down using PXR-siRNA compared to non-specific SCR-siRNA (Figure 6A). A statistically significant (∼80%, P < 0.05) decrease was also observed in the basal expression of CYP4F12. While ∼30% decrease was observed in the basal expression of UGT1A5, this was not statistically significant when compared to hepatocytes transfected with the non-specific SCR-siRNA. To confirm the specificity of the PXR-siRNA we also determined whether basal expression levels of the housekeeping genes 18S and GAPDH were affected. We did not observe a statistically significant decrease in the expression of these genes (data not shown) following PXR-siRNA transfection.Figure 6.

Bottom Line: Genes, both previously known to be involved and not known to be involved in drug disposition, with PXR response elements (PXREs) located upstream, within or downstream from their potentially associated genes, were identified.We observed promoter-dependent patterns with respect to the binding of various coactivators and corepressors involved in the regulation of CYP4F12, CYP3A4, CYP2B6, UGT1A1 and P-glycoprotein.In conclusion, our findings indicate that PXR is involved in the regulation of CYP4F12 and that PXR along with SRC1 binds to a broad range of promoters but that many of these are not inducible by rifampicin.

View Article: PubMed Central - PubMed

Affiliation: Department of Drug Metabolism and Pharmacokinetics, Merck & Co, Rahway, NJ 07065, USA. niresh_hariparsad@merck.com

ABSTRACT
Chromatin immunoprecipitation (ChIP) studies were conducted in human hepatocytes treated with rifampicin in order to identify new pregnane-X receptor (PXR) target genes. Genes, both previously known to be involved and not known to be involved in drug disposition, with PXR response elements (PXREs) located upstream, within or downstream from their potentially associated genes, were identified. Validation experiments identified several new drug disposition genes with PXR binding sites. Of these, only CYP4F12 demonstrated increased binding in the presence of rifampicin. The role of PXR in the basal and inductive response of CYP4F12 was confirmed in hepatocytes in which PXR was silenced. We also assessed the association of PXR-coactivators and -corepressors with known and newly identified PXREs. Both PXR and the steroid receptor coactivator (SRC-1) were found to bind to PXREs in the absence of rifampicin, although binding was stronger after rifampicin treatment. We observed promoter-dependent patterns with respect to the binding of various coactivators and corepressors involved in the regulation of CYP4F12, CYP3A4, CYP2B6, UGT1A1 and P-glycoprotein. In conclusion, our findings indicate that PXR is involved in the regulation of CYP4F12 and that PXR along with SRC1 binds to a broad range of promoters but that many of these are not inducible by rifampicin.

Show MeSH
Related in: MedlinePlus